JP5970560B2 - Method for predicting survival time of patients suffering from solid cancer based on B cell density - Google Patents

Description

TECHNICAL FIELD The present invention relates to methods and kits for prognosis of survival time of patients suffering from solid cancer.

BACKGROUND OF THE INVENTION Cancer is a complex disease that involves interactions between the immune system and tumors ¹ . A correlation between a “high” intratumoral and surrounding adaptive immune response in colorectal cancer and a good prognosis has been reported previously. In contrast, the “low” density of T cells correlated with poor prognosis ^2-4 . Indeed, of all the various clinical and histopathological criteria currently available ^5,6 , immune T cell infiltration has been shown to be the most important predictive criterion for survival ^{2,7- 9} . This is also supported by a mouse model of immune surveillance and immunoediting ^10-12 . Recent advances in cellular immunology and tumor biology have guided a new approach to adoptive T cell therapy ¹³ with promising results ¹⁴ . However, there is still a need for other reliable methods that can help physicians to predict cancer outcomes in patients.

SUMMARY OF THE INVENTION The present invention relates to a method for predicting the survival time of a patient suffering from solid cancer comprising the steps consisting of: i) a tumor invasion margin (im) in a tumor tissue sample obtained from said patient Ii) comparing the density with a predetermined reference value, and iii) if the density of B cells at the infiltrating margin of the tumor is higher than the predetermined reference value Providing a poor prognosis if the B cell density at the infiltrating margin of the tumor is lower than a predetermined reference value.

DETAILED DESCRIPTION OF THE INVENTION Applying an integrated analysis, we studied gene expression and cell density from 28 different cell types, including the most innate and adaptive immune cell subpopulations in tumors. The inventors have found a cluster of immune cells associated with a particular tumor stage. Among them, B and T lymphocytes are the most prominent immune cells that are organized in the core network and correlate with tumor progression and favorable prognosis. In particular, we found that patients with high B cell density at the tumor infiltrate have long disease-free survival, whereas patients with low B cell density at the tumor infiltrate are poor. Proven to have a prognosis. The combination of the B cell marker in at least one region of the tumor (im and / or ct) and at least one marker selected from the group consisting of CD3, CD8 and CDR45RO also gives better patient identification It was.

  Accordingly, the present invention relates to a method for predicting the survival time of a patient suffering from solid cancer comprising the steps consisting of: i) a tumor infiltration margin (im) in a tumor tissue sample obtained from said patient Ii) comparing the density with a predetermined reference value, and iii) if the density of B cells at the infiltrating margin of the tumor is higher than the predetermined reference value Providing a poor prognosis if the B cell density at the infiltrating margin of the tumor is lower than a predetermined reference value.

  In one embodiment, the patient suffers from a cancer selected from the group consisting of: adrenocortical cancer, anal cancer, bile duct cancer (eg, peripillar cancer, lower bile duct cancer, intrahepatic bile duct cancer), bladder cancer Bone cancer (eg, osteoblastoma, osteochondroma), hemangioma, chondromyx fibromas, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma , Lymphoma, multiple myeloma), cancer of the brain and central nervous system (eg meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, ganglion) , Schwannoma, germinoma, craniopharyngioma), breast cancer (eg, noninvasive ductal carcinoma, invasive ductal carcinoma, invasive lobular carcinoma, noninvasive lobular carcinoma, gynecomastia), Castleman disease (For example, giant lymph node hyperplasia, vascular alveolar lymph Hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (eg, endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophageal cancer Gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumor (eg choriocarcinoma, destructive choriocarcinoma), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (eg renal cell carcinoma), larynx And hypopharyngeal cancer, liver cancer (eg, hemangioma, hepatic adenoma, localized nodular hyperplasia, hepatocellular carcinoma), lung cancer (eg, small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, Nasal and paranasal sinus cancer (eg, nasal neuroblastoma, median granulomas), nasopharyngeal cancer, neuroblastoma, oral and pharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma Cell tumor, lateral Myoma (eg, fetal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (eg, melanoma, non-melanoma skin cancer), stomach cancer, testicular cancer ( For example, seminoma, non-seminoma germ cell cancer), thymic cancer, thyroid cancer (eg follicular cancer, undifferentiated cancer, poorly differentiated cancer, medullary thyroid cancer, goiter), vaginal cancer, vulvar cancer, and uterine cancer (eg Uterine leiomyosarcoma).

  As used herein, the term “tumor tissue sample” has its general meaning in the art and is removed, including after surgical tumor resection or after collection of a tissue sample for biopsy. Includes small pieces or slices of tissue. The tissue tumor sample shall include the infiltrating margin surrounding the tumor and may or may not include the center of the tumor. As used herein, “invasive margin” has its general meaning in the art and refers to the cellular environment surrounding a tumor. Tumor tissue samples can of course be subjected to various well-known post-harvest preparation and storage techniques (eg, fixation, storage, freezing, etc.) before determining B cell density at the infiltrating margin (im) of the tumor. it can. Typically, tissue tumor samples can be paraffin embedded or frozen.

  The methods of the invention are particularly suitable for disease free survival (DFS) or overall survival (OS).

  As used herein, the term “B cell” has its general meaning in the art and refers to a cell produced in the bone marrow that expresses a membrane-bound antibody specific for an antigen. Following interaction with the antigen, it differentiates into plasma cells that secrete antibodies specific for the antigen or into memory B cells. “B cells” and “B lymphocytes” are used interchangeably. Typically, B cells are characterized by the expression of B cell markers on their cell surface. As used herein, the term “B cell marker” refers to a surface molecule on a B cell that is specific for a particular B cell. B cell markers suitable for use in the present invention include, but are not limited to, surface IgG, kappa and lambda chains, Ig alpha (CD79 alpha), Ig beta (CD79 beta), CD19, B220 (CD45R), CD20 , CD21, CD22, CD23, CD27, or any other CD antigen specific for B cells. Typically, B cells are CD20 + cells.

  Determining the density of B cells at the infiltrating margin of the tumor can be determined by any well known method in the art. Typically, such methods involve contacting a tumor tissue sample with at least one selective binding agent capable of selectively interacting with B cells. The selective binding agent can be a polyclonal or monoclonal antibody, an antibody fragment, a synthetic antibody, or other protein-specific agent (such as a nucleic acid or peptide aptamer). Typically, the selective binding agent binds to any of the B cell markers, such as an antibody specific for any of these molecules. Preferred B cell selective binding agents bind to CD19, CD20, CD21, CD22, or CD37. A particularly preferred B cell selective binding agent binds to CD20. Several antibodies have been described in the prior art, and many antibodies are also commercially available as described in the examples. For detection of antibodies that allow the presence of B cells to be detected by a microscope or automated analysis system, the antibodies are directly tagged with a detectable label (such as an enzyme, chromogen, or fluorescent probe). Alternatively, it may be detected indirectly using a secondary antibody conjugated with a detectable label.

  A preferred method according to the present invention is immunohistochemistry. Typically, a tissue tumor sample is first incubated with a labeled antibody directed against one B cell marker of interest (eg, CD20). After washing, the labeled antibody bound to the B cell marker of interest is revealed by appropriate techniques and is generated by a labeled antibody (eg, radioactive, fluorescent, or enzyme label) depending on the type of label. Multiple labels can be performed simultaneously. Alternatively, the method of the invention may use an amplification system (to enhance the staining signal) and a secondary antibody conjugated to an enzyme molecule. Such conjugated secondary antibodies are commercially available from, for example, Dako (EnVision system). Counterstaining may be used (eg, H & E, DAPI, Hoechst). Other staining methods can be achieved using any suitable method or system (including automatic, semi-automatic, or manual systems) that will be apparent to those skilled in the art.

As used herein, B cell density is counted as the number of these cells counted per unit of tissue sample surface area (eg, counted per cm ² or mm ² of tumor tissue sample surface area). As the number of B cells to be expressed). As used herein, B cell density can also be expressed as the number of B cells per volume unit of sample (eg, as the number of B cells per cm ^{3 of} a tumor tissue sample). As used herein, the density of B cells can also consist of the percentage of B cells per total cell (set to 100%).

The predetermined reference value used for comparison may comprise a “cut-of” value that can be determined as described herein below. Each reference (“cutoff”) value for each biological marker may be predetermined by performing a method comprising the following steps:
a) providing a collection of tumor tissue samples from cancer patients;
b) For each tumor tissue sample provided in step a), provide information related to the actual clinical outcome for the corresponding cancer patient (ie disease-free survival (DFS) or overall survival (OS) duration) about;
c) providing a series of arbitrary quantitative values;
d) for each tumor tissue sample included in the collection provided in step a), determining the B cell density at the infiltrating margin of the tumor;
e) classifying the tumor tissue sample in each of two groups for one specific arbitrary quantitative value provided in step c): (i) from the arbitrary quantitative value included in the series of quantitative values A first group comprising a tissue tumor sample exhibiting a quantitative value for the density that is lower; (ii) a tissue tumor exhibiting a quantitative value for the density that is higher than the arbitrary quantitative value included in the series of quantitative values A second group comprising samples; thereby two groups of tumor tissue samples are obtained for said specific quantification value, wherein the tumor tissue samples of each group are listed separately;
f) the actual clinical outcome in the patient from which (i) the quantitative values obtained in step e) and (ii) the tumor tissue samples contained in the first and second groups defined in step f) are derived. Calculating statistical significance between them;
g) repeating steps f) and g) until all arbitrary quantitative values provided in step d) have been tested;
h) setting the predetermined reference value (“cutoff” value) as if the highest statistical significance (most significant) consists of any quantitative value calculated in step g).

  As disclosed above, the method allows for the setting of a single “cut-off” value that allows discrimination between poor and good prognosis. In practice, as disclosed in the examples herein, it is generally understood that a high statistical significance value (eg, a low P value) is generally over a range of arbitrary quantitative values (single (Not only for any quantitative value). Thus, in one alternative embodiment of the method for determining the “cutoff” value above, optionally setting a minimum statistical significance value (minimum threshold of significance, eg, maximum threshold P value), Any range of quantitative values with a higher statistical significance value calculated in step g) (more significant, eg, lower P value) is retained, thereby providing a range of quantitative values. Said range of quantitative values consists of “cut-off” values according to the invention. According to this particular embodiment of the “cut-off” value, comparing the B cell density determined in step i) with a range of values delimiting said “cut-off” value, the prognosis of a poor or good clinical outcome. Can be determined. In certain embodiments, the cutoff value consisting of a range of quantitative values is from a range of values centered on the quantitative value for which the highest statistical significance value is found (eg, generally the lowest P value found). Become.

  Typically, a given reference value may consist of a B cell density value (eg, CD20 + cell density) per whole cell (set at 100%), which has a poor prognosis (eg, short disease-free survival time). ) And, in contrast, may consist of B cell density values that correlate with a good prognosis (eg, long disease-free survival time).

  In a particular embodiment, the comparison step may comprise a classification of quantitative values measured for cell density in each of the two groups: (i) if the quantitative value for cell density is higher than a predetermined corresponding reference value. A first group called “Hi” and (ii) a second group called “Lo” when the quantitative value for cell density is lower than a predetermined corresponding reference value. From the example, if the result of the comparison step consists of a “Hi” value, then it flows that a good prognosis is provided (FIG. 1). Conversely, if the result of the comparison step consists of a “Lo” value, then a poor prognosis is provided (FIG. 1). The score can also be determined according to Table 1.

  The method of the invention may further comprise the steps of: i) at least one at the infiltrating margin (im) of the tumor and / or at the center of the tumor (ct) in the tumor tissue sample obtained from said patient. Determining the density of further cell types, and ii) comparing said density to a predetermined reference value.

  Typically, additional cell types are selected from the group consisting of T cells or between a specific subset of T cells, including cytotoxic T cells or memory T cells.

  As used herein, the term “T cell” has its general meaning in the art and includes cells within the T cell lineage (thymocytes, immature T cells, mature T cells, and the like). Including). Typically, T cells are characterized by the expression of T cell markers on their cell surface. As used herein, the term “T cell marker” refers to a surface molecule on a T cell that is specific for a particular T cell. Suitable T cell markers for use in the present invention include, but are not limited to, surface CD3, CD4, CD8, CD45RO, or any other CD antigen specific for T cells. Typically, T cells are CD3 + cells.

  As used herein, the term “cytotoxic T cell” has its general meaning in the art and refers to a T cell, once activated by an MHC-antigen complex, Releases the protein perforin, which forms pores in the plasma membrane of the target cell; this causes ions and water to flow into the target cell, causing it to swell and eventually lyse. Cytotoxic T cells also release granzyme, a serine protease that can enter target cells through the perforin pore and induce apoptosis (cell death). Most cytotoxic T cells have the protein CD8 present on the cell surface, which is attracted to parts of class I MHC molecules. Typically, cytotoxic T cells are CD8 + cells.

  As used herein, the term “memory T cells” has its general meaning in the art and is specific for the antigen it encounters first and is sought during a secondary immune response. Refers to a subset of T cells that can be obtained. Memory T cells are characterized by their expression on the cell surface of CDR45RO. Typically, memory T cells are CD45RO + cells.

  In certain embodiments, the methods of the invention may further comprise the steps of: i) determining the density of T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient. And ii) comparing the density with a predetermined reference value.

  In a particular embodiment, the method of the invention may further comprise the steps of: i) determining the density of T cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from said patient. And ii) comparing the density with a predetermined reference value.

  In certain embodiments, the methods of the invention may further comprise the steps of: i) determining the density of T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient. Ii) determining the density of T cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from the patient, and iii) comparing the density to a predetermined reference value.

  In certain embodiments, the methods of the invention may further comprise the step of: determining the density of cytotoxic T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient. And ii) comparing the density with a predetermined reference value.

  In a particular embodiment, the method of the invention may further comprise the step of: determining the density of cytotoxic T cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from said patient And ii) comparing the density with a predetermined reference value.

  In certain embodiments, the methods of the invention may further comprise the steps of: i) determining the density of cytotoxic T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient. Determining, ii) determining the density of cytotoxic T cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from the patient, and iii) comparing the density to a predetermined reference value about.

  In certain embodiments, the methods of the invention may further comprise the step of: determining the density of cytotoxic T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient. And ii) comparing the density with a predetermined reference value.

  In certain embodiments, the methods of the invention may further comprise the step of: determining the density of memory T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient; And ii) comparing the density with a predetermined reference value.

  In certain embodiments, the methods of the invention may further comprise the step of: determining the density of memory T cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from said patient And ii) comparing the density with a predetermined reference value.

  In certain embodiments, the methods of the invention may further comprise the steps of: i) determining the density of memory T cells at the center of the tumor (ct) in a tumor tissue sample obtained from said patient. Ii) determining the density of memory T cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from the patient, and iii) comparing the density to a predetermined reference value.

  For said additional cell density, the comparison step may also include a classification of quantitative values measured for each cell density in each of the two groups: (i) the quantitative value for the cell density is a predetermined corresponding reference value A first group called “Hi” when higher than, and (ii) a second group called “Lo” when the quantitative value for cell density is lower than a predetermined corresponding reference value.

  Scores that are a complex of classifications made for B cell density and for additional densities may also be calculated as depicted in Tables 1-9 to make it easier to understand the results of the comparison process.

  The method of the present invention is more accurate than currently used staging methods (eg, UICC-TNM). Thus, the methods of the invention can be applied to monitor the effectiveness of anticancer treatments. For example, the present invention provides a method for monitoring the effectiveness of treatment of a subject with a drug, comprising the following steps: (i) performing the method according to the present invention to Predicting patient survival time prior to administration, (ii) predicting patient survival time after administering said agent by performing a method according to the invention, (iii) of step a) Comparing the survival time with the survival time of step b) and iv) Conclusion that the drug is effective for the treatment of cancer if the survival time of step b) is higher than the survival time of step a) To provide. In the case where the conclusion is negative, the physician can then adapt the treatment by prescribing different dosages or by prescribing another drug to be administered. The methods of the invention may also be particularly suitable for determining whether a patient is considered a responder to a treatment (eg, an immunotherapeutic agent). Typically, a patient can be eligible for treatment if a good prognosis is provided by the methods of the invention. The methods of the invention may also be particularly suitable for determining whether adjuvant treatment (eg, chemotherapy) is required. For example, if a good prognosis is provided by the methods of the present invention, subsequent anti-cancer treatment may not include any adjuvant chemotherapy. However, if a poor prognosis is provided by the methods of the present invention, then the patient may be eligible for adjuvant chemotherapy.

  The present invention includes a kit for carrying out the method of the present invention comprising the means for determining the cell density described above. For example, a kit according to the present invention may comprise one or a combination or set of antibodies, each type of antibody being specifically directed to one cell type. Suitable means include antibodies, antibody derivatives, antibody fragments, and the like. The kits of the present invention can optionally include additional components useful for performing the methods of the present invention. By way of example, a kit can include body fluid (eg, buffer), one or more sample compartments, instructional materials that describe the performance of the methods of the invention, and the like.

  The invention is further illustrated by the following figures and examples. However, these examples and drawings should not be construed in any way as limiting the scope of the invention.

The Kaplan-Meier curve illustrates the duration of disease-free survival according to CD20 cell density at the infiltrating margin (im) of the tumor (test cohort, n = 107). Cut-off values for cell density were defined by the cohort's optimal p-value. The Kaplan-Meier curve illustrates the persistence of disease-free survival according to the CD20 cell density at the infiltrating margin (im) of the tumor in combination with the CD8 cell density at the center of the tumor (ct) (test cohort, n = 107 ). Cut-off values for the density of CD20 and CD8 cells were defined by the cohort's optimal p-value. The Kaplan-Meier curve illustrates the persistence of disease-free survival according to the CD20 cell density at the tumor invasive margin (im) in combination with the CD8 cell density at the tumor invasive margin (im) (test cohort, n = 107). Cut-off values for the density of CD20 and CD8 cells were defined by the cohort's optimal p-value. The Kaplan-Meier curve illustrates the persistence of disease-free survival according to the CD20 cell density at the tumor infiltrating margin (im) in combination with the CD3 cell density at the center of the tumor (ct) (test cohort, n = 107 ). Cut-off values for the density of CD20 and CD3 cells were defined by the cohort's optimal p-value. The Kaplan-Meier curve illustrates the duration of disease-free survival according to the CD20 cell density at the tumor invasive margin (im) in combination with the CD3 cell density at the tumor invasive margin (im) (test cohort, n = 107). Cut-off values for the density of CD20 and CD3 cells were defined by the cohort's optimal p-value. The Kaplan-Meier curve illustrates the persistence of disease-free survival according to the CD20 cell density at the tumor infiltrating margin (im) in combination with the CD45RO cell density at the tumor center (ct) (test cohort, n = 107 ). Cut-off values for the density of CD20 and CD45RO cells were defined by the cohort's optimal p-value. The Kaplan-Meier curve exemplifies the persistence of disease-free survival according to the CD20 cell density at the tumor invasive margin (im) in combination with the CD45RO cell density at the tumor invasive margin (im) (test cohort, n = 107). Cut-off values for the density of CD20 and CD45RO cells were defined by the cohort's optimal p-value. The Kaplan-Meier curve illustrates the persistence of disease-free survival according to the CD20 cell density at the infiltrating margin (im) of the tumor in combination with the CD8 cell density at the center of the tumor (ct) (test cohort, n = 107 ). Cut-off values for the density of CD20 and CD8 cells were defined by the cohort's optimal p-value. Group “het” is a sustained disease-free survival of Hi or Lo CD20 cell density at the infiltrating margin (im) and a combination of Hi or Lo CD8 cells at the center of the tumor (ct) and at the infiltrating margin (im). Is illustrated. Kaplan-Meier curve illustrates the persistence of disease-free survival according to the CD20 cell density at the tumor invasive margin (im) in combination with the CD3 cell density at the tumor center (ct) and at the invasive margin (im) (Test cohort, n = 107). Cut-off values for the density of CD20 and CD3 cells were defined by the cohort's optimal p-value. Group “het” is the sustained disease-free survival of the combination of Hi or Lo CD20 cell density at the infiltrating margin (im) with Hi or Lo CD3 cells at the tumor center (ct) and at the infiltrating margin (im). Is illustrated. Kaplan-Meier curve exemplifies persistence of disease-free survival according to CD20 cell density at the tumor invasive margin (im) in combination with CD45RO cell density at the tumor center (ct) and at the invasive margin (im) (Test cohort, n = 107). Cut-off values for the density of CD20 and CD45RO cells were defined by the cohort's optimal p-value. Group “het” is a sustained disease-free survival of Hi or Lo CD20 cell density at the infiltrating margin (im) and a combination of Hi or Lo CD45RO cells at the tumor center (ct) and at the infiltrating margin (im). Is illustrated. The Kaplan-Meier curve illustrates the duration of disease-free survival according to CD20 cell density at the infiltrating margin (im) of the tumor (validation cohort, n = 415). Cut-off values for cell density were defined by the cohort's optimal p-value.

Example 1:
Materials & methods:
patient:
Patients with colorectal cancer who underwent primary resection at Laennec / HEGP (Hopital Europpeen George Pompidou) Hospital were randomly selected (N = 107). The validation cohort (N = 415) was previously described (Galon J, Costes A, Sanchez-Cabo F, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313 : 1960-4.). Time to recurrence or disease free time was defined as the time period from the date of surgery for the relapsed patient to the date of tumor recurrence confirmed and for the disease free patient from the date of surgery to the date of the last follow-up. A secure web-based database (TME.db) integrated clinical data and data from high-throughput technology (36).

Construction of Tissue Microarray Using tissue microarray equipment (Beecher Instruments, Alphelys, Plaisir, France), we selected two different representative regions of the tumor. Tumor center (ct) and infiltrating margin (im) were punched from paraffin-embedded tissue blocks (0.6 mm and 1 mm diameter, respectively). Tissue microarrays were constructed and cut into 5 μm sections for immunohistochemical staining.

Immunohistochemistry After antigen retrieval and quenching of endogenous peroxidase activity, sections were subjected to antibodies against CD3 (SP7), CD8 (4B11), CD45RO (OPD4), and CD20 (L26; DAKO, Carpinteria, CA). Envision + system (enzyme conjugated polymer backbone conjugated to secondary antibody) and DAB-chromogen were applied (Dako, Copenhagen, Denmark). Tissue sections were counterstained with Harris hematoxylin. Slides were analyzed using an image analysis workstation (SpotBrowser, Alphelys, Plaisir, France). Density was recorded as the number of positive cells per tissue surface area (mm ² ). For each overlap, the average density was used for further statistical analysis.

Statistical analysis Kaplan-Meier curves were used to assess the impact of immune parameters on disease-free survival. The significance of these parameters was calculated using a log rank test. We used a median and “minimum P-value” approach to apply a cut-off based on the disease-free survival of the patient, dividing the patient into Hi and Lo groups. The Wilcoxon rank sum test was used for pairwise comparisons. P <0.05 was considered statistically significant. All analyzes were performed using statistical software R and Statview.

result:
In situ testing was performed using tissue microarrays from the tumor center and infiltrating margin. Immunostaining for B cells (CD20), T cells (CD3), cytotoxic T cells (CD8), and memory T cells (CD45RO) was quantified using a dedicated image analysis workstation. Accurate measurement of immune cell density within the tumor was performed by counting immune cells and measuring tissue surface area. We evaluated disease free survival according to B cell density. The Kaplan-Meier curve exemplifies the prognostic effect of CD20 on patient survival in combination with or without cell (CD3) and cytotoxic T cell (CD8) and memory T cell (CD45RO) density ( 1 to 10). Patients with high CD20 density at the infiltrating margin of the tumor had better disease-free survival than patients with low CD20 density in the central region (FIG. 1). The combination of CD20 and CD3, CD8 and CDR45RO markers defined groups of patients with very different outcomes. For example, patients with CD20 Lo (im) and at least one “Lo” marker selected from the group consisting of CD3, CD8, and CDR45RO in at least one region of the tumor had a dramatic outcome. . In contrast, patients with CD20 Hi (im) and at least one “Hi” marker selected from the group consisting of CD3, CD8, and CDR45RO in at least one region of the tumor had a good outcome ( Table 11). We verified the results by analyzing an independent cohort of 415 colorectal cancer patients by tissue microarray. Similar results were found (Figure 11). Long disease-free survival was observed among patients with tumors that contained a high density of CD20 + cells (Hi CD20 (im)) at the infiltrating margin of the tumor. Patients with at least one “Hi” marker selected from the group consisting of CD3, CD8, and CDR45RO in at least one region of the tumor (im and / or ct) had the best outcome. Similar hazard ratios and P values were found in both cohorts.

Conclusion:
In conclusion, patients with high density B cells at the tumor infiltrating margin had long disease-free survival, whereas patients with low density B cells at the tumor infiltrating margin had a poor prognosis. Had. The combination of this marker with at least one marker selected from the group consisting of CD3, CD8, and CDR45RO in at least one region of the tumor (im and / or ct) also gives better patient identification It was. The immunological score can then be calculated according to Tables 1-10.

References:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure.

Claims (5)

A method for assisting in predicting the survival time of a patient suffering from solid cancer, comprising:
Determining the density of CD20 + B cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from the patient;
One additional cell type selected from CD8 + cytotoxic T cells or CD45RO + memory T cells at the tumor's infiltrating margin (im) and at the center of the tumor (ct) in a tumor tissue sample obtained from the patient Determining the density,
Comparing each density with a given reference value; and a)
a1) the density of CD20 + B cells at the infiltrating margin of the tumor is higher than a predetermined reference value; and a2) the density of the additional cell type at the infiltrating margin (im) of the tumor is higher than a predetermined reference value; And a3) good prognosis if the density of the further cell type at the center of the tumor (ct) is higher than a predetermined reference value;
b)
b1) the density of CD20 + B cells at the infiltrating margin of the tumor is higher than a predetermined reference value; and b2) the density of the additional cell type at the infiltrating margin (im) of the tumor is lower than a predetermined reference value; And b3) the density of the additional cell type at the center of the tumor (ct) is higher than a predetermined reference value; or b′1) the density of CD20 + B cells at the invasion border of the tumor is higher than a predetermined reference value And b′2) the density of the additional cell type at the infiltrating margin (im) of the tumor is higher than a predetermined reference value; and b′3) the additional cell type at the center of the tumor (ct) The density of is lower than a predetermined reference value;
Or b ″ 1) the density of B cells at the invasive margin of the tumor is lower than a predetermined reference value; and b ″ 2) the density of the additional cell type at the invasive margin (im) of the tumor And b ″ 3) the density of the additional cell type at the center of the tumor (ct) is higher than a predetermined reference value;
Or bi) the density of B cells at the invasive margin of the tumor is higher than a predetermined reference value; and bii) the density of the additional cell type at the invasive margin (im) of the tumor is lower than a predetermined reference value And biii) the density of the additional cell type at the center of the tumor (ct) is lower than a predetermined reference value;
Or b'i) the density of B cells at the infiltrating margin of the tumor is lower than the predetermined reference value; and b'ii) the density of the additional cell type at the infiltrating margin (im) of the tumor is the predetermined reference Lower than the value; and b'iii) the density of the additional cell type at the center of the tumor (ct) is higher than the predetermined reference value;
Or b ″ i) the density of B cells at the invasive margin of the tumor is lower than a predetermined reference value; and b ″ ii) the density of the additional cell type at the invasive margin (im) of the tumor And b '' iii) moderate prognosis when the density of the additional cell type at the center of the tumor (ct) is lower than a predetermined reference value;
c)
c1) the density of B cells at the infiltrating margin of the tumor is lower than a predetermined reference value;
c2) the density of the additional cell type at the infiltrating margin (im) of the tumor is lower than a predetermined reference value; and c3) the density of the additional cell type at the center of the tumor (ct) is a predetermined reference value A method comprising providing a poor prognosis if lower.   2. The method of claim 1, wherein the additional cell type is CD8 + cytotoxic cells.   2. The method of claim 1, wherein the additional cell type is a CD45RO memory T cell.   The method according to any one of claims 1 to 3, wherein the solid cancer is colorectal cancer. A method for assisting in predicting the survival time of a patient suffering from colorectal cancer comprising:
i) determining the density of CD20 + B cells at the infiltrating margin (im) of the tumor in a tumor tissue sample obtained from the patient;
ii) comparing the density with a predetermined reference value; and iii) a good prognosis if the density of B cells at the tumor's invasive margin is higher than the predetermined reference value; and at the tumor's invasive margin Providing a poor prognosis when the density of B cells is lower than a predetermined reference value.

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